Northern blotting technique

Summary

This video tutorial from Shos Biology explores Northern blotting, a technique for detecting specific RNA sequences within a cell. It emphasizes the similarity between Northern and Southern blotting, explaining that the former focuses on RNA while the latter targets DNA. The process involves RNA extraction, denaturing to linearize the molecules, separation using agarose gel electrophoresis, and transfer onto a membrane for hybridization with a complementary DNA probe. The technique is crucial for identifying specific RNA fragments, and the video promises further insights into related blotting methods.

Takeaways

• 📚 Northern blotting is a technique used to detect specific RNA sequences within a mixture of RNA.
• 🔍 It is similar to Southern blotting, which detects DNA, and both involve hybridization techniques.
• 🕵️‍♂️ The name 'Northern blotting' was derived from 'Southern blotting' and does not relate to geographical directions.
• 🧬 The process involves three main stages: extraction of RNA, separation using denaturing gel electrophoresis, and hybridization with a complementary DNA probe.
• 🧪 RNA is extracted from the cell and then broken down into smaller fragments to eliminate complex secondary structures.
• 🔬 Denaturing gel electrophoresis is used to resolve the secondary structure of RNA into a linear form for accurate separation based on length.
• 🧴 Formaldehyde is added to the gel to help resolve the secondary structure of RNA into a linear form for proper separation.
• 📝 Northern blotting uses a different type of membrane for transferring RNA from the gel compared to Southern blotting, often using aminooxy methy filter paper for better RNA binding.
• 💧 The transfer of RNA from the gel to the membrane is facilitated by capillary action, with the setup including a buffer, sponge, gel, membrane, and paper towels.
• 🔦 A radioactive or chemiluminescent probe is used to bind to the specific target RNA, allowing for the detection and identification of the RNA of interest.
• 📉 The final step involves developing an autoradiograph or using a detection method that reveals the location of the target RNA within the membrane.

Q & A

What is the main purpose of Northern blotting?

Northern blotting is a technique used to detect specific RNA sequences within a mixture of RNA extracted from cells.

What is the relationship between Southern blotting and Northern blotting?

Southern blotting and Northern blotting are similar techniques, with the main difference being that Southern blotting is used for detecting DNA, while Northern blotting is used for detecting RNA.

Who discovered the Southern blotting technique?

The Southern blotting technique was discovered by Edwin Southern in 1975.

What is the significance of using formaldehyde in the gel during Northern blotting?

Formaldehyde is used in the gel to help resolve the secondary structure of RNA into a linear form, which is necessary for accurate separation and detection of RNA fragments.

Why is aminooxy methy paper preferred over nitrocellulose paper in Northern blotting?

Aminooxy methy paper has a greater binding affinity towards RNA compared to nitrocellulose paper, making it a better medium for transferring and detecting RNA during Northern blotting.

What is the role of the probe in Northern blotting?

The probe in Northern blotting is a complementary DNA strand that binds specifically to the target RNA sequence. It is often labeled with a radioactive or chemiluminescent molecule to facilitate the detection of the target RNA.

How does the denaturing gel electrophoresis process help in Northern blotting?

Denaturing gel electrophoresis helps to break down the complex secondary structures of RNA into linear forms, allowing for the separation of RNA fragments based on their length.

What is the purpose of the buffer in the blotting setup?

The buffer in the blotting setup facilitates the capillary action that is necessary for transferring the RNA from the gel to the membrane or filter paper.

Why is the RNA transferred from the gel to a membrane or filter paper?

The RNA is transferred to a membrane or filter paper because agarose gel is fragile and not suitable for the probing process, which requires the application of various solutions and buffers.

What is the final step in detecting the target RNA after the transfer to the membrane?

The final step involves adding the probe to the membrane, which binds specifically to the target RNA. The detection is then achieved through the signal given by the radioactive or chemiluminescent label on the probe.

Why is DNA typically used as a probe in Northern blotting instead of RNA?

DNA is typically used as a probe in Northern blotting because it has better interaction with RNA and is more stable than RNA, which can easily degrade. The DNA probe is designed based on the complementary RNA structure.

Outlines

00:00

🧬 Introduction to Northern Blotting

This paragraph introduces the concept of Northern blotting, a technique used to detect specific RNA sequences within a mixture. It emphasizes the importance of understanding Southern blotting as a basis for Northern blotting due to their similarities. The speaker recommends watching a previous video on Southern blotting for those unfamiliar with blotting techniques. Northern blotting was developed to detect RNA, in contrast to Southern blotting, which detects DNA. The process involves three main stages: preparing the target RNA, using hybridization to find a specific sequence, and employing a probe that can bind to the target RNA. The speaker explains the use of complementary DNA strands as probes, which can be labeled with radioactive or chemical markers to identify the target RNA's position in a gel after separation.

05:01

🔬 Denaturing Gel Electrophoresis in Northern Blotting

The second paragraph delves into the specifics of breaking down the secondary structures of RNA into a linear form, which is essential for nucleic acid probing and hybridization. The speaker describes the use of denaturing gel electrophoresis to resolve these structures and separate the RNA fragments based on their length using agarose gel electrophoresis. The paragraph also explains the importance of using formaldehyde in the gel to linearize the RNA and the process of transferring the separated RNA from the gel to a more robust medium, such as aminooxy methy paper, which has a higher binding affinity for RNA than nitrocellulose paper. This transfer process, known as blotting, is critical for the subsequent probing steps.

10:02

🌱 Capillary Action in Blotting Techniques

This paragraph explains the process of transferring RNA from the agarose gel to the membrane filter using capillary action, a technique common to both Northern and Southern blotting. The speaker details the blotting setup, which includes a buffer, sponge, gel, membrane, and paper towels, and describes how the buffer flows through the layers due to the applied weight. The RNA fragments are pushed from the gel onto the membrane, where they adhere due to the filter paper's impermeability to RNA. The buffer continues to move through the membrane, leaving the RNA fragments in place. This process is essential for creating an exact imprint of the RNA fragments on the membrane, which is then ready for probing with a specific probe.

15:02

🔎 Hybridization and Detection of Target RNA

The final paragraph focuses on the hybridization process and the detection of the target RNA using a specific probe. The speaker clarifies that DNA is commonly used as a probe for RNA due to better interaction properties. Once the RNA fragments are transferred to the membrane, the probe, which can be labeled with radioactive isotopes or chemiluminescent molecules, binds specifically to the target RNA. This binding results in a visual signal, such as light or color, which can be captured on X-ray film to produce an autoradiograph. The autoradiograph reveals the presence and size of the target RNA fragment, allowing researchers to identify the specific RNA of interest. The speaker concludes by encouraging viewers to like, subscribe, and share the video, hinting at upcoming Western blotting videos.

Keywords

💡Northern Blotting

Northern blotting is a molecular biology technique used to detect specific RNA sequences among a mixture of RNA molecules. It is named after its discoverer, Edwin Southern, and is analogous to Southern blotting, which is used for DNA. In the context of the video, Northern blotting is the main theme, explaining its process and significance in detecting RNA sequences of interest, such as messenger RNA (mRNA).

💡Southern Blotting

Southern blotting is a technique for transferring DNA from an agarose gel to a membrane, facilitating the detection of specific DNA sequences. It is foundational to understanding Northern blotting, as the latter is a variation of the former adapted for RNA. The video script mentions Southern blotting to provide a basis for understanding the modifications made in Northern blotting.

💡Hybridization

Hybridization in molecular biology refers to the process where two complementary strands of nucleic acids (DNA or RNA) bind together to form a double-stranded molecule. In the script, hybridization is key to Northern blotting, where a complementary DNA probe is used to find a specific RNA sequence by binding to it.

💡Messenger RNA (mRNA)

Messenger RNA, or mRNA, is a type of RNA that carries genetic information from DNA to the ribosome, where proteins are synthesized. In the context of the video, Northern blotting is often used to detect specific mRNA sequences within a cell's RNA mixture.

💡Denaturing Gel Electrophoresis

Denaturing gel electrophoresis is a technique used to separate nucleic acid molecules based on size by disrupting their secondary structure, allowing them to move through a gel matrix. In the script, this process is essential for Northern blotting to linearize RNA molecules before transferring them to a membrane.

💡Formaldehyde

Formaldehyde is a chemical compound used in Northern blotting to help resolve the secondary structure of RNA into a linear form. This is crucial for proper separation of RNA during gel electrophoresis, as mentioned in the script.

💡Agarose Gel

Agarose gel is a type of gel used in electrophoresis to separate DNA, RNA, or proteins based on size. In the video script, agarose gel is used to separate RNA fragments before transferring them to a membrane for Northern blotting.

💡Nitrocellulose Filter Paper

Nitrocellulose filter paper is a type of membrane used in blotting techniques to immobilize nucleic acids or proteins. Although the script mentions its use in Southern blotting, it notes that aminooxy methy paper is preferred for Northern blotting due to its higher binding affinity for RNA.

💡Aminooxy Methy Paper

Aminooxy methy paper is a type of membrane used in Northern blotting that has a greater binding affinity for RNA compared to nitrocellulose filter paper. The script explains that this paper is used to transfer RNA from the agarose gel to the membrane for detection.

💡Probe

A probe in molecular biology is a molecule, often a DNA or RNA sequence, that is used to detect and locate a specific nucleic acid sequence. In the script, a DNA probe complementary to the target RNA sequence is used in Northern blotting to identify the presence of specific mRNA.

💡Autoradiography

Autoradiography is a process used to visualize the location of a radioactively labeled molecule on a membrane or filter. In the context of the video, autoradiography is used after hybridization in Northern blotting to detect the location of the target RNA by the signal emitted from the radioactive or chemiluminescent probe.

Highlights

Introduction to Northern blotting as a technique for detecting RNA, similar to Southern blotting for DNA.

Explanation of the necessity to understand Southern blotting before learning about Northern blotting.

Historical context of blotting techniques, starting with Southern blotting in 1975 and Northern blotting in 1979.

Description of the three major stages in the blotting process: preparation, separation, and detection.

Extraction of RNA as the first step in Northern blotting, focusing on messenger RNA (mRNA).

Use of nucleic acid hybridization to identify a specific sequence of RNA from a mixture.

Generation of complementary DNA strands to serve as probes for RNA detection.

Attachment of radioactive or chemiluminescent molecules to probes for detection purposes.

Utilization of denaturing gel electrophoresis to resolve RNA secondary structures into linear forms.

Difference in gel composition for Northern blotting, including the addition of formaldehyde to linearize RNA.

Transfer of RNA from the gel to a membrane using capillary action in the blotting process.

Use of aminooxy methy paper instead of Nitro cellulose for better RNA binding in Northern blotting.

Setup of the blotting apparatus, including buffer, sponge, gel, membrane, and paper towels.

Process of capillary action transferring RNA from the gel to the membrane filter paper.

Identification of specific RNA fragments of interest using a radioactive or chemiluminescent probe.

Differences between Northern and Southern blotting, including the type of nucleic acid and probe used.

Practical applications of Northern blotting in identifying specific RNA sequences.

Invitation to like, subscribe, and share the video for upcoming Western blotting content.

Transcripts

[Music]

welcome back friends welcome to another

video tutorial from shos biology in this

video lecture we'll be talking about

Northern blotting okay now before

understanding Northern blotting it's

very important that you know Southern

blotting or at least to any of the

blotting techniques because Northern

blotting is a very very similar with

Southern blotting technique so if you

don't know about the southern blotting

technique yet I I'll recommend you to

watch my Southern blotting video which

the description is provided and the link

is provided in the description as well

as uh the link I'll try to put here in

The annotation watch that video first

because there is small modifications of

the Southern blotting in case of

Northern blotting okay so let's see

blotting is a process of

detecting any macro molecules that we

deal with like DNA RNA or proteins if

it's a process of detecting DNA we call

it a southern blotting if it's a process

of detecting RNA we call it Northern

blotting because the names like with the

directions but actually it's nothing to

deal with the directions exactly uh the

first type of blotting technique that

was discovered in

1975 by em Southern based on the name of

the discoverer it was Southern blotting

but later in s in in 1979 4 years later

another technique was discovered uh that

is known as a northern blotting because

you know Southern blotting is a process

of detecting DNA in 1975 it was

discovered in 1979 they discovered the

process of detecting RNA so RNA and DNA

very closely linked so earlier it was

Southern blotting that's why they name

it as a northern blotting when it detect

into the RNA now what is the process of

Northern blotting it's very very similar

that of the Southern blotting in any

blotting process there are three major

stages the first stage is to prepare the

Target the Target in this case of

Northern blotting is RNA so for example

RNA is present inside the cell all the

time in the cytosol of the cell or

inside the nucleus so what we can do

here we simply extract the content of

the cell that should contain RNA

especially we do the process of this

Northern blotting in case of messenger

RNA or mRNA

and the content of mRNA is present

inside the cytool so we take that RNA

out we extract that part of the RNA so

we have a RNA mixture which is a whole

RNA mixture of the cell but again like

any other detection techniques or

blocking techniques the idea is to find

out a specific Target of the RNA which

we know the sequence by the way so how

would you find out a known sequence of

the RNA from a mixture of other rnas the

only way to do that is is do the

hybridization technique or nuclic acid

hybridization because you know nuclic

acid has a very important feature for

example if I tell you the target RNA

that we are looking for has the sequence

a a u u a g g c u a for example this is

the RNA sequence that we want to find

out okay we know that DNA and RNA both

have a compliment feature so if they

find any complimentary strand they are

going to attach to it the complimentary

strand of this RNA will be you know if

we if we uh get this strand in based on

RNA sequence it will be u u a a u c c g

a u if we think of this complimentary

stand as a DNA fragment it could be t t

a a t c c g g a

t so these are the compliment DNA

strands that we have to generate so once

we generate the strands we can put the

Strand as a probe and that can bind to

the Target DNA now if we attach this

this strands this target strands along

with some sort of radio leled or radio

Radioactive molecule or if we attach

with any sort of chemical or enzyme

molecule which is is giving us light

after a reaction we can accurately

identify the position of the target RNA

in the mixture of RNA while we separate

it in the

gel remember that that's what we do in

the northern blotting so the first step

what we do is we extract all the RNA

then we break those rnas down in smaller

fragments because you know rnas tend to

present in complex secondary structure

forms because they are single stranded

so so they they can have the ability to

pair with wherever they find a

complimentary region so RNA never

presents like a linear strands like that

RNA always present in the very complex

structures and secondary structures

formed right we want linear structure of

the RNA and the fragments of linear

structure of the RNA for the process of

nucleic acid probing or nuclic acid

hybridization then only we can find out

the specific sequence of the RNA now if

you look at

here so how can we break this secondary

structure of the RNA down into linear

form so in that case we need to use a

denaturing gel electroforesis process

which will help us to resolve the

secondary structure into linear RNA then

we can separate that RNA using

electroforesis which is an agar Ro gel

electroforesis then we use the probe and

then the process will be done so this

whole stages will be known as the

northern blotting so first what we do so

extraction is done we have a secondary

structure of the

RNA secondary structure of RNA so what

we do now we load this RNA onto agaro

gel let's say this is the agaro gel that

we are talking

about say this is this this drawings are

from the previous lecture that is

Southern blotting which is you you'll

see that very very similar so what we do

we load this thing we load this

secondary structure of the RNA in the

agaro gel now in the agaro gel when we

load them you know agaro gel that we use

here in case of sou Northern blotting is

different compared to the agaro gel we

used in case of Southern blotting in

this agos gel we have

extra formal

deide we add formal deide in the gel

why because this formal deide will help

to resolve the secondary structure into

linear form of the RNA and that is very

very important otherwise uh the RNA will

not be separated based on their length

the idea of using agos gel is to

separate nuclic acid contents based on

their length because agaro is a polymer

of small Matrix and they'll form small

pores inside so smaller length RNA or

DNA fragment can migrate further along

this gel while the larger fragments the

longer fragments will migrate Less in

the gel with the help of this we can

identify where the larger fragment of

the RNA is present where the smaller

fragment of the RN is present but if the

RNA is in secondary structure format

they will not follow this rule so we

need to resolve the structure into

linear form we use formal deide to do

that formal deide makes it linear and

also little bit heat that helps it to

teat and then the RNA will migrate so

once the the aaro gel electroforesis is

complete so we have the bands or the

pattern of where exactly the different

fragments of the RNA present we get this

idea as a band in the agaro gel but the

question is agaro gel is fragile we

cannot use agaro gel for the probing

process because for the probing we need

to apply different Solutions and buffers

weight and all these things that will

not be enough for uh for holding all

those tension by this agoros gel so it's

a better idea to transfer the content of

the gel into a

paper that is usually in case of

Southern blotting was a Nitro cellulose

filter paper now in case of this

Northern blotting we can also use Nitro

cellulose filter paper but it was found

out that nitr cellulose paper is not

working that well in case of Northern

blotting in this case we use aminooxy

methy paper or aminooxy methy filter

paper instead of a Nitro cellulose

filter paper because you know uh at the

beginning we use Nitro cellulose paper

the results are different but then

slowly in search for a better uh

transfer medium we use aminooxy methy

filter paper it's kind of a very simple

that of the Nitro paper or any nylon

paper but it has more affinity and

greater binding Affinity towards the RNA

so what we do now we take this this gel

we take the gel we put it here so now we

do the process of imprinting or

transferring the content of RNA from the

agaro gel to the filter paper okay and

that is when the plotting term comes in

blotting it's the imprinting of a gel

material onto the paper

and that's why we have this whole

process and this blotting technique of

northern and southern is very very

similar because both this technique uses

the pro the the the process of capillary

action for transferring the content of

DNA or RNA from the gel to the membrane

filter now let's look at here in this

total blotting setup in the blotting

setup we have a buffer we have a of a

buffer that that is uh that that will

contain all the mainly it's it's water

containing buffer mostly but we cannot

use plain water for any biological

processes we buffer here and then on top

of buffer we put sponge the sponge helps

there because the water will move sponge

will help in the capillary activity and

up on the top of the sponge we have the

gel so let me let me just highlight the

gel this is the

gel agaro

gel okay and on top of the gel we put

the aminooxy

methy we put the membrane or filter let

me write it down here this is

the the RNA uh filter and we have this

gel the

sponge and on so look at here the gel

and the filter paper are placed on top

of each other first gel then on top that

we have the filter and on the top top of

filter we put some paper towel okay and

then on top of that we put some weight

so this setup is kind of same in case of

Southern blotting as well as Northern

blotting okay now the idea is the the

those buffer will flow based on the

capillary activity from the bottom

towards the top as we apply some weight

from the top according to the capillary

flow water can flow against the

gravitation and it will flow from the

bottom towards the top so as the water

is moving it moves first through the

sponge reaches the gel it passes through

the gel as it's passing through the gel

it is creating Force to all those RNA

fragments that are present there so it

will be pushed and the RNA fragments are

now released from the gel and then they

will migrate towards this filter paper

and once this RNA fragments will go and

attach to the filter paper there is no

way those RNA can migrate because filter

paper is not permeable to RNA molecules

so RNA will be stagnant it will be

attached to the filter paper there while

buffer solution can easily pass through

the membrane and it will reach the paper

towels that's why you put a lot of paper

towel stack there over so that in a

sense of how exactly we push the

fragments of the RNA and and apply it to

attach to the membrane filter once it's

attached to the membrane then the

process is done we exclude everything

out we take the membrane out now the

membrane will have exact imprint of all

those fragments of the RNA that were

present in the gel so now we take that

membrane and remember one thing

importantly because RNA is single

stranded so all these rnas are present

in the in the in the filter paper then

what we do we add the probe which is

designed to go and bind exactly with the

target RNA only the probe is attached

with with radioactive or radiel isotope

or it can be attached with any chem

luminence molecule in either way this

probe is going and bind only with a

specific

Target and that is going to give us

either light or any color or radio if

it's radio leveled isotope then you put

a Xtra film on top of that and after

developing the Xtra film what we'll get

let's say the small version if I draw

this small version of that we will only

find one b and where the target DNA was

present so you'll see by looking at this

gel or or Auto radiogram we can easily

tell which RNA fragment carry our D RNA

of interest in this case let's say this

one if we go back to the gel we can say

yes this is the fragment that carries

the target DNA of our interest so that

is how the target RNA of our interest

sorry that is how the northern blotting

is conducted and it's very similar that

of the Southern blotting the difference

is that in this case we need to use

formal deide in the gel while we're

separating another difference is that uh

in this case of a southern blotting DNA

were attached to the membrane so we need

to denature this another strand to make

it a single strand but in case of RNA we

don't have to do that remember in case

of RNA hybridization or Northern

blotting we need to use we mostly used

DNA as a probe not RNA because RNA RNA

interactions are not that good DNA

interaction with RNA is mostly done so

the the probe with the DNA is simply RNA

DNA

hybrid that we use but in case of

Southern blotting we use DNA DNA

hybridization now the DNA that we

produce here is called complimentary DNA

because it's designed uh by looking at

the complimentary RNA structure okay so

that in a sense is a difference between

southern blotting and western uh

Northern blotting okay so I hope you

understand the video If you like this

video please hit the like button

subscribe to my channel because Western

blotting videos are going to come and

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thank you